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Results for “Animals, Genetically Modified”

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At least 19 recordsLinked to original sources

PubMed2013

Delivery of recombinant adeno-associated virus vectors to rat diaphragm muscle via direct intramuscular injection.

The diaphragm is the most important inspiratory muscle in all mammals, and ventilatory insufficiency caused by diaphragm dysfunction is the leading cause of morbidity and mortality in many genetic and acquired diseases affecting skeletal muscle. Currently, pharmacological inhibitors, genetically modified animals, and invasive procedures are used to study disorders affecting the diaphragm. However, these methodologies can be problematic because of off-target drug effects and the possible nonphysiological consequences of lifelong genetic alterations. Therefore, alternative methods to study this important respiratory muscle are needed. To resolve this, we have developed a methodology to deliver recombinant adeno-associated virus (rAAV) vectors to the rat diaphragm via direct intramuscular injection. We hypothesized that by direct injection of rAAV into the muscle we can selectively target the diaphragm and establish a novel experimental method for studying signaling pathways and also provide a strategy for effectively using rAAV to protect the diaphragm against disease. This report describes the methods and evidence to support the use of rAAV as a therapeutic intervention to study rat diaphragm biology during conditions that promote diaphragm dysfunction.

Animals↗

PubMed2025

An advanced cytosine base editor enabled the generation of cattle with a stop codon in the β-lactoglobulin gene.

β-Lactoglobulin (BLG) is an allergen present in milk that can induce an acute immune response in certain individuals. The successful use of cytosine base editors (CBEs) can introduce stop codons into premature mRNA, thereby generating animals with disrupted genes that negatively regulate target traits. In this study, we employed a CBE system to target the major milk allergen BLG in bovine embryos, mammary epithelial cells, and live cattle. First, the precise single-base editing of the BLG gene in bovine embryos was achieved by designing an effective sgRNA to induce a c.61C > T substitution in the coding region, converting codon 21Gln (p.21Gln) to a premature stop codon. Sanger sequencing revealed an editing efficiency of 83.3% (20 out of 24 embryos), including two homozygous edits. Second, a bovine mammary epithelial cell line harboring BLG edits was constructed using the same CBE system. Sequencing showed that the designed sgRNA1 enabled the simultaneous conversion of three consecutive cytosines (c.59-61CCC > TTT) to thymines. At position c.61, single-cell clones exhibited monoallelic or biallelic editing (BLGc.61C > T), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLGc.61C > T mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLGc.61C > T single-base-edited dairy cow that despite its heterozygosity, showed significantly reduced BLG expression in the mammary epithelial cells and milk. Collectively, this study demonstrates the feasibility of using CBEs to disrupt BLG expression in dairy cows and provides a foundation for application in generating hypoallergenic dairy products.

Animals↗

PubMed2018

Using mouse transgenic and human stem cell technologies to model genetic mutations associated with schizophrenia and autism.

Solid progress has occurred over the last decade in our understanding of the molecular genetic basis of neurodevelopmental disorders, and of schizophrenia and autism in particular. Although the genetic architecture of both disorders is far more complex than previously imagined, many key loci have at last been identified. This has allowed in vivo and in vitro technologies to be refined to model specific high-penetrant genetic loci involved in both disorders. Using the DISC1/NDE1 and CYFIP1/EIF4E loci as exemplars, we explore the opportunities and challenges of using animal models and human-induced pluripotent stem cell technologies to further understand/treat and potentially reverse the worst consequences of these debilitating disorders.This article is part of a discussion meeting issue 'Of mice and mental health: facilitating dialogue between basic and clinical neuroscientists'.

Animals↗

PubMed2025

Innovations in Transgene Integration Analysis: A Comprehensive Review of Enrichment and Sequencing Strategies in Biotechnology.

Understanding the integration of transgene DNA (T-DNA) in transgenic crops, animals, and clinical applications is paramount for ensuring the stability and expression of inserted genes, which directly influence desired traits and therapeutic outcomes. Analyzing T-DNA integration patterns is essential for identifying potential unintended effects and evaluating the safety and environmental implications of genetically modified organisms (GMOs). This knowledge is crucial for regulatory compliance and fostering public trust in biotechnology by demonstrating transparency in genetic modifications. This review highlights recent advancements in T-DNA integration analysis, specifically focusing on targeted DNA enrichment and sequencing strategies. We examine key technologies, such as polymerase chain reaction (PCR)-based methods, hybridization capture, RNA/DNA-guided endonuclease-mediated enrichment, and high-throughput resequencing, emphasizing their contributions to enhancing precision and efficiency in transgene integration analysis. We discuss the principles, applications, and recent developments in these techniques, underscoring their critical role in advancing biotechnological products. Additionally, we address the existing challenges and future directions in the field, offering a comprehensive overview of how innovative DNA-targeted enrichment and sequencing strategies are reshaping biotechnology and genomics.

Transgenes↗

PubMed2026

Hypoimmune platforms: from rejection to immune evasion and regulatory implications.

The growing gap between organ demand and clinical availability has renewed interest in immune-evasive graft strategies, yet rejection and lifelong immunosuppression remain major barriers to durable success. Advances in genome editing enable immune-evasive cell platforms designed to avoid immune recognition while replacing missing function in allogeneic settings. This review summarizes current strategies for engineering immune-evasive grafts that simultaneously suppress adaptive and innate immune responses. We discuss how coordinated modulation of antigen presentation and immune checkpoint pathways can protect transplanted allogeneic cells and tissues from T, NK, and macrophage-mediated rejection. We also present the emerging concept of integrating hypoimmune engineering with genetically modified porcine donors, where extensive genome editing has reduced, but not eliminated, xenogeneic immune barriers. Combining donor genome modification with immune-evasive graft design represents a promising conceptual advance toward xenograft survival, though whether full elimination of systemic immunosuppression is achievable remains to be established clinically. We further examine how the regulatory landscape for these products is evolving across major jurisdictions, and how differences in approval pathways, manufacturing standards, and long-term surveillance requirements shape the path to clinical translation. Finally, we outline the safety considerations and remaining limitations in immune evasion that must be addressed to enable clinical implementation.

Graft Rejection↗

PubMed2025

Abiotic conditions can modify the penetrance of transgene-based lethality systems for insect population control.

Modern genetic biocontrol techniques for insect pest management, when compared to chemical insecticide spraying, offer high species specificity and reduced environmental impact, and some of these methods require the environmental release of genetically modified (GM) insects. Because organisms exposed to different environments often show variability in phenotype and gene expression, it is likely that GM insects will also experience environmentally mediated variation, potentially compromising pest control efficiency. This study examines the impact of temperature and nutrition on the early embryonic Tet-off conditional lethality system in Drosophila melanogaster. By independently manipulating parental and offspring environments, we assessed how exposure to variable environments influenced the probability of larval hatching and the transcript abundance of the transgenic system. Our findings revealed that: (i) transgene performance distinctly responds to temperature and nutrition; (ii) thermal stress has a greater impact when embryos, rather than parents, are exposed; and (iii) extreme nutritional conditions can markedly reduce the penetrance of transgenic lethality. Although changes in transgene transcript abundance were observed across environments, these changes did not fully explain the phenotypic variation, suggesting that factors downstream of transcription probably drive variation in transgenic lethality.

Animals↗

PubMed2024

Identifying transgene insertions in Caenorhabditis elegans genomes with Oxford Nanopore sequencing.

Genetically modified organisms are commonly used in disease research and agriculture but the precise genomic alterations underlying transgenic mutations are often unknown. The position and characteristics of transgenes, including the number of independent insertions, influences the expression of both transgenic and wild-type sequences. We used long-read, Oxford Nanopore Technologies (ONT) to sequence and assemble two transgenic strains of Caenorhabditis elegans commonly used in the research of neurodegenerative diseases: BY250 (pPdat-1::GFP) and UA44 (GFP and human α-synuclein), a model for Parkinson's research. After scaffolding to the reference, the final assembled sequences were ∼102 Mb with N50s of 17.9 Mb and 18.0 Mb, respectively, and L90s of six contiguous sequences, representing chromosome-level assemblies. Each of the assembled sequences contained more than 99.2% of the Nematoda BUSCO genes found in the C. elegans reference and 99.5% of the annotated C. elegans reference protein-coding genes. We identified the locations of the transgene insertions and confirmed that all transgene sequences were inserted in intergenic regions, leaving the organismal gene content intact. The transgenic C. elegans genomes presented here will be a valuable resource for Parkinson's research as well as other neurodegenerative diseases. Our work demonstrates that long-read sequencing is a fast, cost-effective way to assemble genome sequences and characterize mutant lines and strains.

Caenorhabditis elegans↗

PubMed2025

Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.

CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.

Animals↗

PubMed2025

Comprehensive transcriptomic analysis of myostatin-knockout pigs: insights into muscle growth and lipid metabolism.

Pigs are a vital source of protein worldwide, contributing approximately 43% of global meat production. Recent genetic advancements in the myostatin (MSTN) gene have facilitated the development of double-muscling traits in livestock. In this study, we investigate the transcriptomic profiles of second-generation MSTN-knockout (MSTN-/-) pigs, generated through CRISPR/Cas9 gene editing and somatic cell nuclear transfer (SCNT). Using RNA sequencing, we compared the transcriptomic landscapes of muscle tissues from MSTN-/- pigs and wild-type (WT) counterparts. The sequencing yielded an average unique read mapping rate of 86.7% to the Sus scrofa reference genome. Our analysis revealed 15,142 differentially expressed genes (DEGs), including 121 novel genes, with 2554 genes upregulated and 1629 downregulated in the MSTN-/- group relative to the wild-type group. Notable transcriptomic changes were identified in genes associated with muscle development, lipid metabolism, and other physiological processes. These findings provide valuable insights into the molecular consequences of MSTN inactivation, with potential applications in the optimization of livestock breeding and advancements in biomedical research.

Animals↗

PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗

PubMed2026

Emerging trends in genome editing of wild animals.

Globally, nearly one million species are currently threatened with extinction, highlighting the need for more efficient solutions to biological conservation. Genome editing, which allows for faster and more precise changes in genomes, is a promising technique for boosting populations through facilitated adaptation, management of invasive or pathogenic populations, and potentially even facilitating the revival of extinct species. These approaches belong to a new field of research termed conservation biotechnology, which places a great responsibility on researchers and decision makers to ensure sustainability. In this paper, we have mapped the emerging trends in genome editing of wild animals. Current projects primarily focus on population control and de-extinction, with fewer initiatives aimed at preserving threatened species. We then explore four critical dimensions of conservation biotechnology: the technology itself, new perspectives on conservation practices, research organization, and governance and policy. Despite its potential, key questions remain-particularly whether genome editing can increase genetic diversity without causing unintended non-target impacts. Genome editing also provokes new perspectives on conservation practices where ecosystem-wide impact assessment, case-by-case evaluations, and post-release monitoring needs to be prioritized. Furthermore, conservation biotechnology is heavily funded through private funding showing varying stakeholder interest, which can lead to untraditional and less transparent research processes. Stakeholders, including local and indigenous people, are only to a certain degree involved, which may weaken inclusion of local knowledge and monitoring efforts. Finally, concerning governance and policy, there is an urgent need to develop more adequate regulation of conservation biotechnology, as environmental release of genome-edited animals challenges definitions and guidelines in current nature protection laws and GMO regulations. Based on our analysis, we outline key points for further investigation toward a more sustainable approach to conservation biotechnology.

Animals↗

PubMed2026

Generation of germline-transmitting transgenic sheep by piggyBac-mediated transgenesis using pronuclear and cytoplasmic gene injection approaches.

Sheep represent an important large-animal model for biomedical research and biopharmaceutical production. Although the piggyBac transposon system offers efficient and stable genomic integration, the optimal gene delivery strategy for ovine embryos remains unclear. This study evaluated piggyBac-mediated transgenesis using pronuclear injection (PNI) in both in vivo- and in vitro-derived embryos and assessed cytoplasmic injection (CTI) as an alternative approach. In vivo-derived embryos were obtained from superovulated Kıvırcık ewes approximately 40 h after gonadotropin-releasing hormone administration, whereas in vitro-derived embryos were produced from slaughterhouse-derived oocytes. All embryos were injected with the hyperactive piggyBac transposase-based pmhyGENIE-3 construct (10 ng/µL). In vivo-derived embryos were transferred immediately after injection, whereas in vitro-derived embryos were cultured for 3 days and screened for EGFP expression prior to transfer. Among 65 in vitro-derived embryos injected by PNI, no transgenic offspring was obtained. In contrast, PNI of 19 in vivo-derived embryos resulted in one transgenic lamb (5.3%). CTI of 12 in vivo-derived embryos similarly produced one transgenic lamb (8.3%). Whole-genome sequencing of the healthy founder male produced by CTI identified a single detectable genomic integration locus on chromosome 10 within a non-coding RNA locus (LOC121820439). Germline transmission was confirmed by in vitro fertilization using sperm from the founder male, with EGFP expression detected in 10.1% (10/99) resulting embryos. These findings provide proof-of-concept evidence for piggyBac-mediated transgenesis in sheep and support the feasibility of cytoplasmic injection as an alternative gene-delivery approach under the conditions tested.

Animals↗

PubMed2026

Evaluation of germline transmission of electroporation-mediated double gene-edited cattle lines.

Gene editing in livestock using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) offers a promising approach for genetic improvement in cattle. This study evaluated germline transmission and mutation stability of double-knockout cattle generated by zygote electroporation. Previously reported myostatin/beta-lactoglobulin (MSTN/BLG) and newly generated α-1,3-galactosyltransferase (GGTA1/BLG) double-knockout cattle were produced using CRISPR/Cas9-mediated genome editing. Targeted deep sequencing demonstrated extensive somatic mosaicism across multiple tissues. Computer-assisted sperm analysis (CASA) demonstrated normal sperm motility in MSTN/BLG double-knockout males. Fertilization of wild-type oocytes produced heterozygous embryos, with mutation frequencies of 37.76 ± 10.74% at the MSTN locus and 54.80 ± 7.73% at the BLG locus, as assessed by T7 endonuclease I (T7E1) assay. MSTN/BLG double-knockout sperm were subsequently used for embryo production and for artificial insemination of GGTA1/BLG double-knockout females. Healthy offspring were successfully obtained (n = 3), alongside one stillborn calf. Targeted deep sequencing of all four progenies revealed highly variable allele frequencies that deviated substantially from the approximately 50% expected for heterozygous germline transmission. In contrast, whole-genome sequencing (WGS) results were consistent with Mendelian expectations, underscoring the limitations of PCR-based targeted sequencing for assessing germline transmission in mosaic founders. These results show that CRISPR/Cas9-edited embryos generated by electroporation can develop into healthy, sexually mature cattle capable of germline transmission. While variable transmission rates were observed owing to founder mosaicism, non-mosaic F1 offspring were successfully generated. However, direct, embryo-mediated gene-editing strategies remain technically and economically challenging for large-scale commercial calf production, and reports in cattle are limited. This study provides a reference for future applications of gene-edited embryos and their germline propagation.

Animals↗

PubMed2026

Hemizygous loss-of-function variants of EIF1AX are associated with a syndromic neurodevelopmental disorder.

Pathogenic variants of genes encoding initiation factors can cause neurological diseases, including neurodevelopmental disorders and brain abnormalities. The eukaryotic translation initiation factor 1 A, X-linked (EIF1AX) is a gene located at Xp22.12 that plays an important role in the regulation of translation initiation. Here, we identified de novo hemizygous EIF1AX variants in male individuals with neurodevelopmental disorders and explored their possible involvement in these neurological disorders. We performed trio-based exome or whole genome sequencing in four families. The pathogenicity of EIF1AX variants was evaluated using a molecular dynamic simulation and transgenic Drosophila models. We identified four de novo hemizygous EIF1AX variants in four male individuals with variable neurodevelopmental delay, dysmorphic features, behavioral problems, ophthalmological abnormalities, and structural abnormalities in the brain. One variant was predicted to cause a splicing alteration, and minigene analysis confirmed exon skipping leading to the generation of a premature termination codon. In transgenic Drosophila harboring wild-type (WT) EIF1AX or the three other EIF1AX missense variants, overexpression of WT and the p.(Asn17Asp) variant caused structural abnormalities in the compound eye, whereas the p.(Lys64Glu) and p.(Asp90Gly) variants significantly reduced these eye abnormalities. In addition, WT overexpression resulted in significant axonal toxicity in the Drosophila optic nerve, causing a significant reduction in the number of axons, whereas all mutants showed only a mild reduction in axonal number. Our findings indicated that all variants resulted in different degrees of EIF1AX loss-of-function. Overall, EIF1AX is a novel gene for which loss-of-function variants appear to produce syndromic neurodevelopmental disorders in males.

Humans↗

PubMed2026

Immune profiling in a living human recipient of a gene-edited pig kidney.

Xenotransplantation of gene-edited pig kidneys offers a promising solution to the shortage of kidneys for organ transplantation. We recently performed a gene-edited pig kidney transplantation into a living human recipient with end-stage kidney disease. Here, using transcriptomics, proteomics, metabolomics and multiplexed imaging, we conducted high-dimensional immune profiling in this individual. Despite profound depletion of circulating T cells, early T cell-mediated rejection occurred within 1 week after transplantation, likely driven by subtherapeutic immunosuppression and the presence of residual CD8+ T cells in lymph nodes. This T cell-mediated rejection event was reversed by intensified immunosuppression. After treatment, adaptive immunity remained suppressed, whereas innate immune activation, characterized by sustained monocyte and macrophage activity along with elevated levels of interleukin-1 beta and granulocyte-macrophage colony-stimulating factor, persisted. Comparative transcriptomic analysis showed that xenograft rejection profiles resembled those typically observed in human allograft rejection, while also revealing unique innate immune signatures. We did not detect antibody-mediated rejection. The levels of circulating pig donor-derived cell-free DNA rose during the initial rejection episode and declined with treatment, supporting the potential of cell-free DNA measurements as a noninvasive biomarker of xenograft rejection. These findings define the distinct immune landscape of kidney xenotransplantation and highlight the need for regimens targeting both innate and adaptive immunity to improve outcomes.

Animals↗

PubMed2026

Bacterial polar metabolites modulate β-amyloid toxicity and cholinergic dysfunction in models of Alzheimer's disease.

Alzheimer's disease is characterized by progressive neurodegeneration driven by β-amyloid (Aβ) toxicity, oxidative stress, and cholinergic dysfunction. In this study, we investigated whether polar metabolites derived from a cultivable bacterial isolate could modulate Aβ-associated neurodegenerative phenotypes in complementary experimental models. A bioactivity-guided approach identified an aqueous fraction with high antioxidant capacity in DPPH, FRAP, and ORAC assays. In a transgenic Drosophila melanogaster model expressing human Aβ, treatment with this fraction significantly reduced amyloid accumulation and attenuated neurodegenerative histopathological alterations. In human SH-SY5Y neuronal cultures, the metabolites improved cell viability under therapeutic, but not preventive, conditions following exposure to aggregated Aβ. The aqueous fraction also exhibited significant inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Whole-genome sequencing assigned the bioactive isolate to the genus Providencia, with comparative genomic analyses suggesting its placement within a distinct taxonomic lineage. Metabolomic profiling by LC-ESI-MS/MS revealed a diverse set of polar metabolites, including metabolites putatively annotated based on spectral matching, previously associated with neuroprotective and cholinesterase-modulating activities. Collectively, these findings demonstrate that bacterial polar metabolites can modulate key pathological features of Alzheimer's disease, supporting their relevance for mechanistic studies of Aβ toxicity and cholinergic dysfunction.

Animals↗

PubMed2024

Misregulation of bromotyrosine compromises fertility in male Drosophila.

Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. Here, we report detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.

Animals↗

PubMed2026

Generation of full-length wild-type and mutated futsch transgenes in Drosophila-efficient Gibson Assembly of ultra-large cDNAs.

Drosophila Futsch is a key microtubule-associated protein (fly homolog of MAP1B) that regulates microtubule organization, synaptic terminal growth, and neuronal development. Functional analysis of futsch has long been limited by the inability to clone and express a full-length futsch transgene, owing to its exceptional size (~16.5 kb) and extensive repetitive sequences. Here, I present an efficient and reproducible method for cloning both wild-type and mutated full-length Drosophila futsch cDNA (16,488 bp) using Gibson Assembly. These resulting cDNAs were used to generate UAS-futsch transgenes. When expressed in neurons, the wild‑type transgenic Futsch associated with microtubule and rescued the synaptic morphological defects observed in futschK68 mutants. This approach substantially reduces the time and complexity compared with traditional cloning techniques. Furthermore, I highlight common pitfalls encountered during the cloning process and provide practical solutions to enhance cloning efficiency. This protocol offers a broadly applicable and cost-effective framework for cloning otherwise intractable large cDNAs from Drosophila and other organisms.

Animals↗